Alpha-Amylase

=Introduction= Discovered and isolated by Anselme Payen in 1833, amylase was the first enzyme to be discovered. Amylases are hydrolases, acting on α-1,4-glycosidic bonds. They can be further subdivided into α,β and γ amylases. α-Amylase is an enzyme that acts as a catalyst for the hydrolysis of alpha-linked polysaccharides into α-anomeric products. The enzyme can be derived from a variety of sources, each with different characteristics. α-Amylase found within the human body serves as the enzyme active in pancreatic juice and salvia. α-Amylase is not only essential in human physiology but has a number of important biotechnological functions in various processing industries.

=Structure = Shown as 1hvx is the structure of the thermostable α-amylase of Bacillus stearothermophilus (BSTA). BSTA is comprised of a single polypeptide chain. This chain is folded into three domains: A, B and C. These domains are generally found on all α-amylase enzymes. The A domain constitutes the core structure, with a (β/α)8-barrel.The  B domain consists of a sheet of four anti-parallel β-strands with a pair of anti-parallel β-strands. Long loops are observed between the β-strands. Located within the B domain is the binding site for Ca2+-Na+-Ca2+. Domain C consisting of eight β-strands is assembled into a globular unit forming a Greek key motif. It also holds the third Ca2+ binding site in association with domain A. Positioned on the C-terminal side of the β-strands of the (β/α)8-barrel in domain A is the active site. The catalytic residues involved for the BSTA active site are Asp234, Glu264, and Asp331. The residues are identical to other α-amylases, yet there are positional differences which reflect the flexible nature of catalytic resides. CaII and CaI with Na found in the interior of domain B and CaIII at the interface of domain A and C, constitute the metal ion binding sites. All α-amylases contain one strongly conserved Ca2+ ion for structural integrity and enzymatic activity. CaI is consistent in α-amylases, however there are structural differences between the linear trio of CaI, CaII and Na in other enzymes. CaIII acts as a bridge between two loops, one from α6 of domain A, and one between β1 and β2 of domain C.

Chloride Dependent Enzymes
A family of chloride-dependent enzymes, including salivary and pancreatic α-amylase, require the binding of a chloride ion to be allosterically activated. The function of the chloride ion still remains uncertain. No relationship has been observed between the anion binding affinity and its activity, indicating the complexity between the binding parameters and mechanism it activates. Studies have shown that nitrite and nitrate ions with pancreatic α-amylase fit within the chloride binding site, thus making all the necessary hydrogen bonds and enhancing the relative activity by 5-fold.

=Function=

Mechanism
In the human body, α-amylase is part of digestion with the breakdown of carbohydrates in the diet. The mechanism involved includes catalyzing substrate hydrolysis by a double replacement mechanism, forming a covalent glycosyl-enzyme intermediate and hydrolyzed through oxocarbenium ion-like transition states. One of the carboxylic acids in the active site acts as the catalytic nucleophile during the formation of the intermediate. A second carboxylic acid operates as the acid/base catalyst, supporting the stabilization of the transition states during the hydrolysis.

Human Salivary and Pancreatic α-Amylase
Salivary α-Amylase hydrolyzes the (α1-4) glycosidic linkages of starch, separating it into short polysaccharide fragments. Once the enzyme reaches the stomach, it becomes inactivated due to the acidic pH. Further breakdown of starch occurs by secretion of a second form of the enzyme by the pancreas. Pancreatic juice enters the duodenum and pancreatic α-amylase further cleaves starch to yield maltose, maltotriose and oligosaccharides. The oligosaccharides are referred to as dextrins, which are fragments of amylopectin consisting of (α1-6)branch points. Microvilli of the intestinal epithelia break maltose and dextrins into glucose, which gets absorbed into the circulatory system. Glycogen has a relatively similar structure as starch, and thus proceeds in the same digestive pathway.

Regulation
α-Amylase is regulated through a number of inhibitors. These inhibitors are classified according to six categories, based on their tertiary structures. Inhibitors of α-amylase block the active site of the enzyme. In animals, inhibitors control the conversion of starch to simple sugars during glucose peaks after a meal so that breakdown of glucose occurs at a rate the body can handle. This is particularly important for diabetics, who require low quantities of α-amylase to maintain control over glucose levels. After taking insulin however, pancreatic α-amylase escalates. Plants use these inhibitors as a defense mechanism to inhibit the use of α-amylase in insects, thus protecting themselves from herbivory.

=Industrial Uses= α-Amylase is used extensively in various industrial processes. In textile weaving, starch is added for warping. After weaving, the starch is removed by Bacillus subtilis α-amylase. Dextrin, which is a viscosity improver, filler, or ingredient of food, is manufactured by the liquefaction of starch by bacteria α-amylase. Bacterial α-amylases of B.subtilis, or B.licheniformis are used for the initial starch liquefaction in producing high conversion glucose syrup. Pancreatitis can be tested by determining the level of amylases in the blood, a result of damaged amylase-producing cells, or excretion due to renal failure. α-Amylase is used for the production of malt, as the enzyme is produced during the germination of cereal grains.

Alpha-amylase
3ij7, 1xv8, 1c8q, 1bsi, 1smd, 1hny – hAAM – human

1xgz, 1q4n, 1kb3, 1kbb, 1kbk, 1kgu, 1kgw, 1kgx, 1jxj, 1jxk, 2cpu - hAAM (mutant) 3n8t – TkAAM – Thermococcus kodakarensis

3k8k – BtAAM – Bacterioides thetaiotaomicron

3kwx, 2guy – AoAAM – Aspergilluys oryzae

2wpg – AAM – Xanthomonas campestris

2wc7, 2wcs, 2wkg – AAM catalytic region – Cyanobacterium

3bh4 – BaAAM – Bacillus amyloliquefaciens

1e3x, 1e43 – BaAAM chimera

1ht6, 1amy - bAAM – barley

3bsg – bAAM (mutant) 3dhu – AAM – Lactobacillus plantarum

3dc0 – AAM – Bacillus KR8104

3bcf, 1wza – HoAAM – Halothermothrix orenii

2die, 1wp6 – AAM alkaline – Bacillus sp.

1ud2, 1ud4, 1ud5, 1ud6, 1ud8 - AAM – ''Bacillus sp. KSM-K38''

1ud3 – AAM (mutant) – ''Bacillus sp. KSM-K38''

2gjr – BhAAM – Bacillus halmapalus

2b5d – AAM – Thermotoga maritima

2c3g, 2c3v – BhaloAAM – Bacillus halodurans

1ji1, 1ji2, 1bvz - TvAAM – Thermoactinomyces vulgaris

1wzk, 1wzl, 1wzm, 1izj, 1izk, 1jf5, 1jf6 – TvAAM (mutant) 1mwo, 1mxd – PwAAM – Pyrococcus woesei

1ob0, 1bli - BlAAM (mutant) – Bacillus licheniformis 1vjs – BlAAM precursor

1b0i, 1aqm, 1aqh – PhAAM - Pseudoalteromonas haloplanktis

1jd7, 1jd9 - PhAAM (mutant) 1g5a – NpAAM – Neisseria polysaccharea

1hvx - BaAAM – Bacillus stearothermophilus

1qho – GsAAM – Geobacillus stearothermophilus

1jae – TmAAM – Tenebrio molitor

1pif – pAAM – pig

6taa, 2aaa - AoAAM

AAM binary complexes

1xd0, 1xd1, 1cpu, 1jfh - hAAM + saccharide

1b2y, 1xcw, 1xcx - hAAM + acarbose

3blk, 3blp, 1z32, 1nm9, 1mfu, 1mfv, 3cpu - hAAM (mutant) + saccharide

3dhp, 1xh0, 1xh2 - hAAM (mutant) + acarbose

3ij8, 3ij9 – hAAM catalytic intermediate

2qmk, 3bai – hAAM + NO2

3baw – hAAM + N3

3bax - hAAM (mutant) + N3

3bak – hAAM (mutant) + NO3

1xh1 - hAAM (mutant) + Cl

2qv4, 3baj, 3bay - hAAM + acarbose + NO2

3old, 3ole, 3olg, 3oli – hAAM + statin

1u2y, 1u30, 1u33 – hAAM + inhibitor 3n92, 3n98 – TkAAM + saccharide

3l2l, 3l2m, 1vah, 1wo2, 1ua3, 1pig, 1ppi - pAAM + saccharide

1hx0 – pAAM + acarbose

1kxq, 1kxt, 1kxv – pAAM + antibody VHH fragment

1bvn, 1dhk – pAAM + protein inhibitor

1ose – pAAM + acarbose

3k8l - BtAAM (mutant) + saccharide

3k8m - BtAAM + acarbose

2d2o, 1jl8, 1jib - TvAAM + saccharide

3a6o – TvAAM + acarbose

2d0f, 2d0g, 2d0h, 1vb9, 1vfm, 1vfo, 1vfu, 1uh2, 1uh4 - TvAAM (mutant) + saccharide

1uh3 - TvAAM (mutant) + acarbose

1ava – bAAM + protein inhibitor

1bg9, 1rpk - bAAM + acarbose

1p6w – bAAM + substrate analog 1rp8, 1b1y, 1rp9 - bAAM (mutant) + saccharide

3bsh, 2qpu, 2qps - bAAM (mutant) + acarbose

3bcd - HoAAM + saccharide

3bc9 - HoAAM + acarbose

2gjp, 1w9x - BhAAM + saccharide

2gvy - AoAAM + saccharide

1zs2, 1s46, 1mvy, 1mw0, 1mw1, 1mw2, 1mw3 - NpAAM (mutant) + saccharide

1bag - BsAAM + saccharide – Bacillus subtilis

1ua7 – BsAAM + acarbose

2d3l, 2d3n - BacAAM + saccharide – Bacillus

2c3h, 2c3w, 2c3x - BhaloAAM + saccharide

1mxg - PwAAM + acarbose

1g9h, 1g94 - PhAAM + saccharide

1kxh - PhAAM (mutant) + acarbose

1l0p – PhAAM + NO3

1e40 – BaAAM chimera + saccharide

1e3z - BaAAM chimera + acarbose

1fa2 - AAM + saccharide – Sweet potato

1qhp - GsAAM + saccharide

1clv, 1viw, 1tmq – TmAAM + protein inhibitor 1gah, 1gai – AaAAM + acarbose – Aspergillus awamori

3gly, 1agm, 1glm – AaAAM + saccharide

Pullulanase alpha-amylase
2wan – AAM – Bacillus acidopullululyticus

2fgz – KaAAM – Klebsiella aerogenes

2e8y - BsAAM

Pullulanase alpha-amylase binary complexes

2fh6, 2fh8, 2fhb, 2fhc, 2fhf - KaAAM + saccharide

3fax - AAM + saccharide – Streptococcus agalactiae

2e8z, 2e9b - BsAAM + saccharide

1g1y - TvAAM (mutant) + saccharide

Beta-amylase
2xfr – bBAM 1wdp – sBAM – soybean

2dqx, 1uko, 1ukp – sBAM (mutant)

1vem, 5bca, 1cqy, 1b90 – BcBAM – Bacillus cereus

1ven - BcBAM (mutant)

Beta-amylase binary complexes

2xff – bBAM + acarbose

2xfy, 2xg9, 2xgb, 2xgi – bBAM + inhibitor

1wdq, 1wdr, 1wds, 1v3h, 1v3i, 1q6d, 1q6e, 1q6f, 1q6g - sBAM (mutant) + saccharide

1q6c, 1bfn, 1bya, 1byb, 1byc, 1byd, 1btc - sBAM + saccharide

1j0y, 1j0z, 1j10, 1j11, 1j12, 1j18, 1b9z - BcBAM + saccharide

1veo, 1vep, 1itc - BcBAM (mutant) + saccharide

Gamma-amylase
1lf6 – TtGAM – Thermoanaerobacterium thermosaccharolyticum

1lf9 - TtGAM + acarbose

Maltohexaose-producing amylase
1wp6 - BacMAM 1wpc – BacMAM + saccharide

Maltogenic amylase
1gvi, 1sma – MAM – Thermus sp.

Taka amylase
2taa – AoTAM

7taa – AoTAM + acarbose

=References=